RU2761637C1 - ESCHERICHIA coli BL21(DE3)/ pET32 v 11-Cre CELL STRAIN PRODUCING SITE-SPECIFIC Cre RECOMBINASE - Google Patents

Abstract

FIELD: genetic engineering.

SUBSTANCE: invention relates to the field of genetic engineering and molecular biology, in particular to the recombinant plasmid pET32v11-Cre, which provides synthesis of the recombinant protein 6His-S-NLS-Cre. The invention also discloses a strain of Escherichia coli BL21(DE3)/pET32v11-Cre cells and a method for obtaining the specified 6His-S-NLS-Cre protein using the specified strain. The plasmid according to the invention provides synthesis of the specified recombinant protein in Escherichia coli cells due to the location of the Cre recombinase gene in the 3’ region relative to the T7 promoter and lac operator and in the 5’ region relative to the T7 terminator and was obtained by cloning the cDNA of Cre recombinase into the pET32v11 vector, so that the cDNA of Cre recombinase with N-terminal NLS was in the same reading frame with 6-histidine and S-epitopes and amino acid sequences of thrombin, enterokinase, and TEV protease cleavage sites. It can be used for the production of recombinant site-specific recombinase Cre.

EFFECT: present invention makes it possible to obtain a catalytically active Cre recombinase with a high yield.

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Description

TECHNICAL FIELD OF THE PRESENT INVENTION

The invention relates to the field of genetic engineering, molecular biology and is a method for producing a strain of a genetically modified organism producing a catalytically active recombinase Cre.

The invention was created with the financial support of the Ministry of Science and Higher Education of the Russian Federation under the Agreement No. 075-15-2019-1661 of October 31, 2019.

Prior art of the present invention

et al., 2004). В модельных системах, в зависимости от взаимной ориентации LoxP сайтов в геноме, в результате действия Cre может происходит вырезание, интеграция, инверсия или транслокация фрагментов ДНК.The system of site-specific recombination Cre-LoxP from bacteriophage P1 infecting E. coli cells has become widespread for genomic engineering and genetic analysis of model organisms, allowing one to study the functions of individual genes or regulatory elements in the context of an organism (Branda and Dymecki, 2004; McLellan et al., 2017; Sauer, 1998). The Cre-LoxP system consists of a Cre tyrosine recombinase and 34 bp recombination sites called LoxP (Sternberg and Hamilton, 1981). The wild-type LoxP site, ATAACTTCGTATAatgtatgcTATACGAAGTTAT, has a similar architecture to the FRT site and consists of two inverted 13 bp repeats. (shown in capital letters), separated by an asymmetric 8 bp spacer. (shown in lowercase). The nucleotide sequence of the spacer determines the orientation of the LoxP site in the genome or genetic constructs. In nature, the function of the Cre-LoxP system is to circulate the P1 phage genome into a plasmid, separate physically linked circular plasmids, and maintain the copy number of such plasmids in E. coli cells (Austin et al., 1981;

et al., 2004). In model systems, depending on the mutual orientation of LoxP sites in the genome, Cre action can result in excision, integration, inversion, or translocation of DNA fragments.

For in vivo manipulation of the mammalian cell gene, the Cre-LoxP system was first used in a cell culture model (Sauer and Henderson, 1988), and subsequently in a transgenic mouse model (Lakso et al., 1992; Orban et al., 1992). By 2017, more than 3000 papers have been published using the Cre-LoxP system (McLellan et al., 2017). The Cre-LoxP system is often used to generate conditional gene knockout mouse models. In this case, a transgenic mouse strain is obtained in which one or more critical gene exons are flanked by LoxP sites (so-called "floxed" mouse strains). To induce a gene knockout, such an animal line is crossed with driver lines of transgenic animals that express Cre recombinase under the control of ubiquitous, tissue-specific or inducible promoters. As a result of Cre action, the genome fragment flanked by LoxP sites is removed, which leads to inactivation of the target gene (McLellan et al., 2017).

To date, more than two thousand driver lines of transgenic mice and many floxed animal lines have been obtained and characterized (McLellan et al., 2017). In addition, reporter lines of mice were obtained, which made it possible to evaluate the effectiveness of Cre in monitoring the expression of reporter genes, such as β-galactosidase or Egfp. Many of these animal lines are available to researchers and deposited in nurseries such as the Jackson laboratory, and information on these lines of mice can be found at https://mice.jax.org. Thus, for almost 30 years since the first application on a transgenic mouse model in 1992, the Cre-LoxP system remains extremely popular and relevant for the genetic analysis of model animals, primarily house mice.

The Cre-LoxP system is also used in genomic engineering RMCE (Recombinase Mediated Cassette Exchange), which was originally developed based on the Flp-FRT site-specific recombination system (Schlake and Bode, 1994). The practical possibility of using Cre-LoxP in RMCE is based on the identification of LoxP sites with mutated spacer sequences, which can recombine only with each other, but not with LoxP sites with other spacer sequences (Siegel et al., 2001). As in the case of the Flp-FRT system, the use of two LoxP sites with different spacers allows one to strictly preserve the orientation of the DNA fragment after recombination, and thus to carry out an exact replacement of the DNA sequence in the genome, flanked by two LoxP sites with different spacers, with the DNA sequence introduced into the cell flanked by the same LoxP sites (Turan et al., 2011; Turan et al., 2013).

et al.). В результате инверсии с использованием одной пары сайтов LoxP прекращается экспрессия первого гена и активируется экспрессия второго гена, после чего с использованием второй пары сайтов LoxP удаляется небольшой фрагмент экспрессионной кассеты. Такая система позволяет осуществлять эффективный мониторинг активности Cre на уровне единичных клеток (

et al.). Технология FLEX находит применение для анализа трансгенных животных. Например, получена линия мышей, несущая в локусе ROSA26 кассету FLEX с Egfp и субъединицей А дифтерийного токсина, которая позволяет за счет Cre-опосредованной рекомбинации активировать экспрессию токсина и тем самым элиминировать определенные популяции клеток в организме мыши (Plummer et al., 2017). Технология Cre-опосредованной рекомбинации с использованием стохастических комбинаций инверсии и удаления фрагментов ДНК была элегантным образом реализована в системе Brainbow для мечения нейронов головного мозга с помощью комбинаций флуоресцентных белков (Cai et al., 2013; Livet et al., 2007).In addition, a technology called FLEX was developed on the basis of the Cre-LoxP system, the application of which is based on the inversion of DNA fragments. In the FLEX system, two reporter genes are flanked by pairs of LoxP sites with different spacers and are oriented so that one gene is under the control of the promoter and is expressed, and the second gene is located in the reverse orientation (

et al.). As a result of inversion using one pair of LoxP sites, the expression of the first gene is stopped and the expression of the second gene is activated, after which a small fragment of the expression cassette is removed using the second pair of LoxP sites. Such a system allows efficient monitoring of Cre activity at the level of single cells (

et al.). FLEX technology finds application in the analysis of transgenic animals. For example, a mouse strain has been obtained that carries a FLEX cassette with Egfp and a diphtheria toxin A subunit at the ROSA26 locus, which allows, through Cre-mediated recombination, to activate toxin expression and thereby eliminate certain cell populations in the mouse body (Plummer et al., 2017). Cre-mediated recombination technology using stochastic combinations of inversion and removal of DNA fragments was elegantly implemented in the Brainbow system for labeling brain neurons using combinations of fluorescent proteins (Cai et al., 2013; Livet et al., 2007).

The advent of genomic editing technology using the CRISPR / Cas9 system made it possible to significantly simplify and accelerate the process of obtaining transgenic animals (Low et al., 2016; Singh et al., 2015), which makes it possible to efficiently obtain both new driver lines of mice with a given tissue-specific expression to recombinase Cre and to produce animal lines carrying LoxP sites in the genome to create a conditioned gene knockout or conduct RMCE. This determines the importance of revising and optimizing genetic analysis methods using site-specific recombination. Currently, DNA free genome editing (DNA free genome editing) technologies are becoming more and more widely used, which, first of all, implies the delivery of genome editing systems into cells in the form of a ribonucleoprotein complex of the Cas9 protein and guide RNA (RNA), and not in the form of expression plasmids. The use of RNPs is more efficient, reduces the risk of modification of off-target genome loci and eliminates the possibility of accidental integration of plasmid DNA encoding Cas9 and guide RNAs into the off-target site of the genome (Metje-Sprink et al., 2019). All these advantages are inherent in the use of site-specific recombinases in the form of proteins. In particular, the first report on the use of the recombinant Cre protein to induce site-specific recombination dates back to 1993 (Baubonis and Sauer, 1993). Alternative methods for the delivery of Cre to mammalian cells involve the use of plasmids or mRNA; however, plasmid DNA can be integrated into a random place in the genome, which requires additional verification of the cell line or model organism for plasmid integration. The use of mRNA site-specific recombinases is quite effective (de Wit et al., 1998; Ponsaerts et al., 2004); however, when mRNA is introduced, time is required for transcription and translation of the protein, which reduces the efficiency of recombination and, in the case of microinjection of mRNA into zygotes, can contribute to the formation of mosaicism. In addition, adenoviruses can be used to express Cre in somatic cells of the body (Akagi et al., 1997). However, the introduction of the virus to experimental animals can induce an immune response, which is unacceptable when studying the function of the genes of the immune system.

In addition, the standard scheme for obtaining animals with conditional knockout involves crossing a line of animals expressing Cre recombinase with a line of animals carrying a genome locus flanked by LoxP sites (McLellan et al., 2017). This has several disadvantages. First, many animal lines expressing Cre were obtained using random transgenesis, and not all such lines were characterized for identification of transgene integration sites and its copy number. Consequently, in this case, the genome of double transgenes with conditional knockout of the target gene will carry randomly integrated DNA fragments with an expression cassette for Cre in one of the chromosomes, which may disrupt the functions of any genes or noncoding RNAs at the site of transgene integration. In addition, there is a lot of evidence of the toxicity of Cre expression for cells (McLellan et al., 2017). When double transgenic animals are obtained in the body of such animals, Cre will be expressed, constitutively or in certain tissues and organs, which can cause a toxic effect, affecting cell proliferation or intracellular signaling pathways or due to nonspecific genome modification at the sites of cryptic LoxP sites.

Thus, the use of the Cre-LoxP site-specific recombination system is one of the important experimental approaches to functional genomics in both mammals and other model organisms such as Drosophila melanogaster (Gurumurthy et al., 2019; McLellan et al., 2017; Sauer , 1998). Considering the widespread use of site-specific recombination systems, including Cre-LoxP, for manipulating the genomes of model organisms or cell lines, it seems relevant to develop and apply approaches based on the use of the recombinant Cre protein. This will increase the efficiency of genomic engineering, expand the arsenal of methods used, eliminate the risk of inappropriate integration of genetic constructs for the expression of Cre into the genome of the recipient cell, and minimize Cre-associated toxicity.

Protein Purification Cre

Bacterial systems for the expression of recombinant proteins are the most accessible and efficient. In nature, Cre recombinase is encoded by the genome of bacteriophage P1, which infects E. coli cells. This property allows efficient production of the recombinant Cre protein in E. coli cells in a catalytically active form without the need for optimization of the codon composition (Van Duyne, 2015).

The main approaches to the purification of the recombinant Cre protein from bacterial expression systems can be divided into methods of purification of Cre in its native form; methods of affinity purification of Cre carrying the corresponding epitopes; methods combining affinity purification and chromatographic methods of native protein purification.

Since the discovery of the Cre-LoxP system, an efficient protocol has been developed for the purification of the native recombinant Cre protein from the bacterial expression system (Abremski and Hoess, 1984). In particular, Cre was purified by chromatography on a phosphocellulose column (often used for the purification of DNA-modifying enzymes (Lu and Erickson, 1997)) and gel filtration on Sephadex G-75 media to obtain a protein with a purity of 98% (Abremski and Hoess, 1984). This protocol was used in subsequent works (Mack et al., 1992), however, in this case, Cre was expressed under the control of the T7 promoter in the E. coli strain BL21 (DE3), which is widely used for the production of recombinant proteins at the present time and has been used in this invention.

Further modification of the methods for the purification of native Cre protein was described in (Ghosh and Van Duyne, 2002). Cre was purified by a combination of two-step cation exchange chromatography on SP and Mono S columns followed by hydroxyapatite chromatography (Ghosh and Van Duyne, 2002). In another work, the native Cre protein for subsequent crystallization was purified using three-stage chromatography on a P11 phosphocellulose column, a Mono S cation exchange column, and gel filtration on a Superdex 200 support (Ennifar et al., 2003).

Alternative methods for purifying Cre are based on the use of different affinity epitopes. For example, Cre was expressed as a fusion protein with MBP (Maltose binding protein), which allowed for the purification of the recombinant protein by affinity chromatography on an amylose column (Kolb and Siddell, 1996). In this work, Cre was modified by adding NLS, the MBP epitope was cleaved with factor Xa protease, and the cleaved MBP protein was removed by ion exchange chromatography on a Q-Sepharose column. Importantly, the N-terminal MBP epitope did not affect the catalytic activity of Cre (Kolb and Siddell, 1996). This Cre purification method was subsequently used to obtain a recombinant protein suitable for microinjection into mouse oocytes (Luckow et al., 2009; Shmerling et al., 2005) or fused with peptides that provide penetration into mammalian cells (Jo et al., 2001) ...

Another variant of Cre affinity purification was based on the use of intein protein elements, which are capable of providing post-translational cleavage of epitopes from the target protein without the use of proteases. Cre was expressed under the control of the T7 promoter as a fusion protein with an intein from Saccharomyces cerevisiae ( Sce VMA intein) and a chitin-binding domain from Bacillus circulans , which provided affinity purification of the recombinant protein using a chitin column (Cantor and Chong, 2001). To activate intein-dependent Cre cleavage, the column with the bound protein was treated with dithiothreitol (DTT). This method made it possible to obtain the Cre protein without any epitopes using only one-step affinity chromatography.

Metal chelate affinity chromatography has found wide application for the purification of Cre from E. coli cells due to its relative simplicity and efficiency (Block et al., 2009). To obtain the recombinant Cre protein using metal chelate affinity chromatography, an expression system from Novagen, plasmid vectors of the pET or pTriEx series, and the corresponding bacterial strains carrying the DE3 lysogen were often used (D'Astolfo et al., 2015; Jo et al., 2001; Martin et al., 2002; Peitz et al., 2002). Alternatively, the Cre protein with a 6 histidine epitope was purified using the QIAexpressionist system (Qiagen) (Santoro and Schultz, 2002).

In addition, glutathione S-transferase (GST) from the organism of Schistosoma japonicum was used as an affinity epitope for the purification of Cre using glutathione sepharose. In particular, GST-Cre was obtained, followed by proteolytic cleavage of the GST epitope with thrombin (Will et al., 2002), or a protein without GST cleavage was used (Jo et al., 2001). The thus obtained protein efficiently penetrated mammalian cells in culture and caused site-specific recombination. In another work, Cre was produced in a bacterial expression system in the form of a protein fusion with GST, which did not affect the ability of Cre to bind to LoxP sites (Kalinichenko et al., 2017) and allowed for specific purification of DNA fragments containing LoxP sites.

As noted above, a number of studies have used a combination of affinity purification and chromatographic methods for purifying the native protein. For example, Cre with an N-terminal 6-histidine epitope was expressed in the pET28b vector (Novagen) and purified by metal chelate affinity chromatography and subsequent cation exchange chromatography. The thus obtained protein was crystallized in complex with oligonucleotide substrates (Martin et al., 2002).

The relevance of obtaining a catalytically active form of Cre from a bacterial expression system is illustrated by the commercial use of such proteins. For example, a recombinant Cre protein derived from E. coli fused to the Tat peptide of the HIV virus and capable of entering cells without the use of special protein transfection reagents is currently commercially available from Merck (https://www.merckmillipore.com /RU/ru/product/TAT-CRE-Recombinase,MM_NF-SCR508?ReferrerURL=https%3A%2F%2Fwww.google.com%2F) and Excellgen (https://www.excellgen.com/stem-cell- reagents-c-13 / cre-recombinase-tatcre-tatnlscre-htnc-htncre-p-52931.html).

Considering the previously published data, obtaining the recombinant Cre protein in a bacterial expression system seems to be the most optimal approach. The Cre protein described in this invention carries NLS and is produced fusion with the N-terminal 6-histidine and S-epitopes, which, if necessary, can be cleaved using the TEV protease. As shown in Fig. 6, the recombinant 6His-S-NLS-Cre protein carrying N-terminal epitopes is active in vitro , which is consistent with the data from (Kolb and Siddell, 1996) that N-terminal epitopes have no effect on Cre activity. The Cre purification method used in this invention is based on a combination of metal chelate affinity chromatography on Ni-NTA agarose and cation exchange chromatography on a Mono S column.

In vitro analysis of Cre recombinase activity

Recombinase Cre is capable of catalyzing the site-specific recombination reaction in vitro . A recombination test using a circular form of plasmid DNA carrying LoxP sites as a substrate was first implemented using an E. coli cell lysate containing the Cre protein (Abremski et al., 1983), and then using a purified recombinant Cre protein ( Abremski and Hoess, 1984). As a substrate in the recombination reaction, circular supercoiled plasmid DNA, linear DNA fragments or circular molecules with nicknames can be used (Abremski et al., 1983). The basic principles of the recombination test have been described by Abremski and colleagues (Abremski and Hoess, 1984). In particular, the Cre reaction buffer included 50 mM Tris pH = 7.5, 33 mM NaCl, 5 mM spermidine, and 500 μg / ml BSA, while spermidine in the reaction buffer can be replaced with 10 mM MgCl ₂ . For the reaction, no energy sources are required, only a saline buffer and 10 mM MgCl ₂ are used. A circular plasmid is used as a substrate, from which, as a result of the action of Cre, two circular reaction products are formed, which, for the convenience of detection, are linearized using restriction endonucleases and analyzed by electrophoresis in an agarose gel. To terminate the reaction, the mixture is heated to + 70 ° C to inactivate Cre.

Since 1984, the in vitro assay for assessing the activity of the recombinant Cre protein has been used in numerous works in different formats with some modifications, most often the protocol and composition of the reaction buffer was based on the work of Abremski and colleagues (Abremski and Hoess, 1984). As a rule, the format of the reaction substrate was varied using circular plasmids, linearized plasmids, short synthetic double-stranded DNA molecules. To quantify the reaction, in some cases, the substrate was radioactively labeled. Kolb and colleagues used two types of reactions: excision of a DNA fragment flanked by LoxP sites from a circular plasmid, and fusion of two circular plasmids carrying one LoxP site. The reaction buffer included 10 mM Tris pH = 7.5, 50 mM NaCl, 10 mM MgCl ₂ and 1 mM DTT (Kolb and Siddell, 1996).

In other works, a linearized plasmid was used as a substrate, from which a DNA fragment is excised by Cre, and a truncated linear fragment and a circular DNA molecule are formed (Cantor and Chong, 2001; Ghosh and Van Duyne, 2002; Santoro and Schultz, 2002). The reaction mixture contained 20 mM Tris pH = 7.5, 300 mM NaCl, 1 mM EDTA, 0.1% β-mercaptoethanol and 0.5 nM substrate and 1000 nM Cre (Santoro and Schultz, 2002). In other works, 50 mM Tris pH = 7.5, 33 mM NaCl, 10 mM MgCl _{2 were used} (Cantor and Chong, 2001); 50 mM Tris, pH = 7.5, 50 mM NaCl, 5 mM spermine hydrochloride, 1 mM EDTA, 1 mM DTT and 100 μg / ml BSA or 50 mM Tris pH 7.5, 50 mM NaCl, 10 mM MgCl _2, and 1 mM DTT ( Ghosh and Van Duyne, 2002). Alternatively, a linear substrate for an in vitro reaction is obtained by PCR (Zhang et al., 2015).

An interesting modification of the in vitro test is based on the use of short double-stranded oligonucleotides carrying LoxP sites that lack one phosphodiester bond on the cut DNA strand on the 3'-side of the cut phosphate residue (Ghosh and Van Duyne, 2002). In this case, as a result of the action of Cre, a stable complex of Cre and the substrate is formed.

In addition, LoxP-Cre in vitro recombination can be effectively used for manipulating recombinant DNA: for manipulating the herpes simplex virus genome (Gage et al., 1992), for obtaining recombinant baculoviruses (Peakman et al., 1992), for inversion open reading frames in plasmids of the FLEX recombination system, being an effective alternative to molecular cloning (Xu and Zhu, 2018).

Recombinant Cre protein is commercially available from NEB under catalog number M0298 (https://international.neb.com/products/m0298-cre-recombinase#Product%20Information), and the recommended reaction buffer is 50 mM Tris-HCl pH = 7.5, 33 mM NaCl, 10 mM MgCl ₂ corresponds to that of (Abremski and Hoess, 1984) and was used in a recent work (Xu and Zhu, 2018). In the present invention , a reaction buffer was used to carry out the in vitro reaction, in accordance with the data from Abremski and colleagues and the recommendations of the NEB company.

Use of the Cre protein for genomic engineering

Recombinase Cre in the form of a protein has been used for genomic engineering for a long time, which is possible due to the rather simple and efficient purification of the recombinant Cre protein (Ghosh and Van Duyne, 2002). Initially, it was shown that lipofection of the Cre protein into a mammalian cell line leads to efficient recombination (integration or excision of a DNA fragment) (Baubonis and Sauer, 1993). It turned out that the MBP-Cre fusion protein has the same catalytic activity in vitro as the native Cre protein, and is able to efficiently induce recombination by electroporation into the mammalian cell line (Kolb and Siddell, 1996).

Araki et al. Demonstrated the principal possibility of efficient Cre-mediated recombination in mouse oocytes after microinjections of circular plasmid DNA expressing Cre (Araki et al., 1995). This approach was also effective for microinjection of Cre mRNA into mouse oocytes (de Wit et al., 1998). Despite the fact that the authors did not find integration into the genome of the plasmid for the expression of Cre (Araki et al., 1995), there is a high probability of integration even of plasmids in circular form. Therefore, it is preferable to use Cre mRNA or protein to manipulate the zygote genome. In particular, Cre-mediated recombination was shown during the implementation of the RMCE scheme upon coinjection of a cassette for the expression of Egfp and the recombinant MBP-Cre protein into oocytes carrying "floxed" cassettes at the β-actin gene locus (Shmerling et al., 2005). Moreover, the injected zygotes were transplanted into pseudopregnant females, producing offspring carrying alleles with correct replacement of expression cassettes. However, in such experiments, the efficiency of cassette replacement using RMCE was rather low, about 0.3% (Shmerling et al., 2005). It is important to note that, unlike plasmids that persist for a long time in cells, the Cre protein is transient in cells, which contributes to a shift in the balance of the RMCE process towards the integration of cassettes into the genome, but not their excision. In another example, microinjection of MBP-Cre protein into mouse oocytes was used to remove marker genes for antibiotic resistance integrated into the genome of embryonic stem cells as part of cassettes for obtaining mice with knockout of the CCR2 and CCR5 genes (Luckow et al., 2009).

Another option for using the recombinant Cre protein is to obtain the so-called "cell-permeable" Cre variants, which penetrate mammalian cells without the use of transfection or electroporation reagents. This is achieved by fusing Cre with certain amino acid sequences that mediate the efficient penetration of the protein into the cell. Such sequences are called membrane translocation sequence, MTS; protein translocation sequence, PTS; protein transduction domain, PTD; cell-penetrating peptide, CPP. This approach was first described using a recombinant Cre protein with an N-terminal 6His epitope and NLS and a C-terminal MTS sequence from the FGF-4 protein (Jo et al., 2001). The 6His-NLS-Cre-MTS protein efficiently induced recombination in model cell lines. Moreover, intraperitoneal administration of the 6His-NLS-Cre-MTS recombinant protein to transgenic mice carrying an inactivated β-galactosidase reporter gene in the ROSA26 locus caused site-specific recombination and activated the expression of the reporter gene in all animal tissues (Jo et al., 2001) ...

Will and colleagues used Cre fused to HIV Tat peptide and HBV PreS2 peptide. These proteins caused efficient recombination in a cell line carrying a reporter construct in the genome (Will et al., 2002). However, it turned out that the Cre protein fused only with NLS from the large T antigen of the SV40 virus, as well as the native Cre protein, also have the ability to penetrate cells and induce recombination (Lin et al., 2004; Will et al., 2002). On cell cultures of fibroblasts and mouse embryonic stem cells, the efficiency of using the 6His-Tat-NLS-Cre protein was confirmed, as well as recombination when using NLS-Cre was shown, and it was found that the MTS sequence from the FGF-4 protein is not necessary for the penetration of Cre into a cell (Peitz et al., 2002). This technology is used to obtain transgenic pigs free of marker genes used for selection of correctly modified fetal fibroblasts for subsequent transfer of nuclei from somatic cells (SCNT) (Kang et al., 2018). In this case, the Cre protein fused with various CPP / PTDs (Tat peptide, R9, and CPP5) was used, among which the CPP5 peptide was the most effective (Kang et al., 2018).

The above examples illustrate various uses of the recombinant Cre protein for genomic engineering in cell lines, mouse oocytes, and adult transgenic mice. Taken together, this indicates the feasibility and importance of obtaining the recombinant Cre protein, and also indicate possible options for its use.

Brief description of the invention

This invention solves the problem of obtaining a bacterial strain that allows you to efficiently produce a recombinant Cre protein fused with 6-histidine and S-epitopes and a nuclear localization sequence from the large T-antigen of the SV40 virus (6His-S-NLS-Cre). A method for purifying the 6His-S-NLS-Cre protein using a combination of metal-chelate affinity and cation-exchange chromatography is described, which makes it possible to obtain catalytically active Cre recombinase with a yield of about 17 μg from 1 ml of culture. The Cre protein can be used for genomic engineering in model organisms and cell lines, as well as for in vitro manipulation of recombinant DNA. In mammalian cells, Cre can be delivered by microinjection, electroporation, or using commercially available protein transfection reagents. Moreover, it is possible to obtain variants of the Cre protein, which actively penetrate into cells. Microinjections of the recombinant Cre protein into the embryos of model organisms carrying LoxP sites in the genome can be used to integrate DNA fragments into the target site of the genome, to remove genomic sequences and obtain a conditioned gene knockout for RMCE. Delivery of the Cre protein can be considered as an alternative to crossing with Cre-expressing driver lines. The use of the Cre protein can increase the efficiency of genomic engineering, expand the arsenal of methods used, and eliminate the risk of inappropriate integration of genetic constructs for the expression of Cre into the genome of the recipient cell.

Implementation / Disclosure of the Present Invention

The invention solves the problem of obtaining a bacterial strain effectively producing the recombinant protein 6His-S-NLS-Cre, which is a site-specific Cre recombinase, modified by adding N-terminal 6-histidine and S-epitopes, which can be used for affinity purification of the recombinant protein (Block et al., 2009; Raines et al., 2000). The recombinant protein produced in the bacterial system is purified using metal chelate affinity chromatography and cation exchange chromatography. The amino acid sequence of the recombinant protein contains cleavage sites for thrombin proteases, enteropeptidase (enterokinase) and TEV (tobacco mosaic virus protease). If necessary, the 6-histidine and S-epitopes are removed by proteolytic cleavage and the Cre protein is obtained, which carries at the N-terminus only the nuclear localization sequence (NLS) from the large T-antigen of the SV40 virus (Kalderon et al., 1984). The presence of NLS is important for the translocation of the Cre protein into the nucleus and at the same time increases the affinity of the Cre protein for cation exchange carriers, which makes the purification of this protein more efficient (more than 90% purity, Fig. 3). In another technical embodiment, the recombinant protein is purified without carrying out proteolytic cleavage of the 6-histidine and S-epitopes, which does not affect the functional activity of the Cre recombinase.

The plasmid vector pET32v11-Cre is obtained using genetic engineering methods by amplifying the Cre recombinase cDNA and subsequent cloning into the pET32v11 vector, so that the Cre cDNA with the N-terminal NLS is in frame with the 6-histidine and S-epitopes and amino acid sequences cleavage sites for thrombin, enterokinase and TEV protease.

Escherichia coli BL21 (DE3) / pET32v11-Cre, producing the 6His-S-NLS-Cre recombinant protein, is obtained by transforming competent cells of Escherichia coli BL21 (DE3) with the plasmid vector pET32v11-Cre. The strain is cultivated on a selective medium containing the antibiotic ampicillin at a concentration of 50 μg / ml, and stored at -80 ° C in an LB medium containing 15% glycerol.

To obtain the 6His-S-NLS-Cre recombinant protein, the cells of the BL21 (DE3) / pET32v11-Cre producer strain are cultivated in a rich TB medium, the induction of recombinant protein synthesis is carried out for 16 hours at a temperature of + 18 ° C by introducing the inductor IPTG into the medium (isopropyl-β-D-1-thiogalactopyranoside) to a final concentration of 0.8 mM. E. coli cell suspension is destroyed in buffer 30 mM HEPES pH = 7.4, 500 mM NaCl, 10 mM KCl, 1 mM MgCl ₂ , 10 mM imidazole, 0.1% NP-40, 5% glycerol, 1 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1x cocktail of protease inhibitors VII (Calbiochem) and the recombinant protein 6His-S-NLS-Cre is purified by metal chelate affinity chromatography on Ni-NTA (Qiagen). The fractions with the maximum Cre concentration are pooled and purified by cation exchange chromatography on a Mono S 5/50 carrier (GE Healthcare). The purified protein is dialyzed against buffer (20 mM sodium acetate pH = 5.0, 40 mM NaCl, 2 mM DTT) and stored in aliquots at -80 ° C. In other embodiments of the method, the recombinant protein is purified using metal chelate affinity chromatography, proteolytic cleavage of the N-terminal epitopes using TEV protease is performed, and the protein is further purified using cation exchange chromatography.

The identity of the purified protein was confirmed by SDS-PAGE and Western blotting.

The specific activity of the purified recombinant protein 6His-S-NLS-Cre is checked using an in vitro recombination test using circular plasmid DNA carrying LoxP sites as a substrate.

The Escherichia coli BL21 (DE3) / pET32v11-Cre cell strain was deposited in the Bioresource Center of the All-Russian Collection of Industrial Microorganisms (BRC VKPM) of the National Research Center "Kurchatov Institute" - GosNIIGenetics under the number B-13908.

Brief Description of Drawings

FIG. 1 shows the genetic map of the pET32v11-Cre plasmid construct encoding the 6His-S-NLS-Cre recombinant protein. The main elements of the plasmid are shown: Ampicillin resistance gene (bla) - gene of resistance to ampicillin (β-lactamase); lacI - lac repressor gene from E. coli ; ColE1 / pBR322 origin - replication origin point; f1 origin - point of origin of replication from bacteriophage f1; 6His-S-NLS-Cre - coding sequence for the recombinant protein 6His-S-NLS-Cre; lac operator - lac operator; T7 promoter - T7 promoter; T7 terminator - T7 terminator; His tag and S-tag - 6 histidine and S-epitopes; thrombin, enterokinase, TEV cleavage site - cleavage sites by the corresponding proteases. In addition, a number of unique restriction sites are shown (EcoRI, SalI, XhoI, PstI, etc.).

FIG. 2 shows the results of SDS-PAGE analysis in 12% acrylamide gel of fractions eluted from a Mono S cation exchange column during purification of the 6His-S-NLS-Cre recombinant protein. Fraction numbers are indicated at the top. A portion of the original cell lysate was dialyzed prior to loading onto a Mono S column. Eluted fractions for such a sample are marked with a “d” at the top. Lane "w" is a sample of the pooled wash fractions and lysate passed through the column. The "inp" lane contains a sample of pooled cell lysate fractions with Cre protein after metal chelate chromatography prior to dialysis. Mw - protein molecular weight marker: PageRuler ™ Prestained Protein Ladder, 10 to 180 kDa (ThermoFisher Scientific). The molecular weight of the marker stripes is indicated to the left of the figure. The band corresponding to the 6His-S-NLS-Cre protein is indicated by an arrow.

FIG. 3 shows the results of analysis in 10% acrylamide gel using SDS-PAGE of the purified recombinant protein 6-His-S-NLS-Cre (lanes "0.85 μg" and "1.7 μg") and samples of cell lysates of the E. coli BL21 strain ( DE3) / pET32v11-Cre before the induction of recombinant protein synthesis (lane "0 h") and after cultivation of the cells of the strain in a medium containing the inducer of protein synthesis IPTG at a concentration of 1 mM for 3 hours at + 37 ° C (lane "3 h" ). To the right is the position of the bands in the Precision Plus Dual Color Protein Standard (# 161-0374 Bio-Rad) protein molecular weight marker. The band corresponding to the 6His-S-NLS-Cre protein is indicated by an arrow.

FIG. 4 shows the results of Western blot analysis of the purified recombinant protein 6-His-S-NLS-Cre (lanes "0.85 μg" and "1.7 μg") and samples of lysates of E. coli BL21 (DE3) / pET32v11-Cre cells before synthesis induction recombinant protein (lane "0 h") and after culturing the cells of the strain in a medium containing the inducer IPTG at a concentration of 1 mM for 3 hours at + 37 ° C (lane "3 h"). For Western blot analysis, murine monoclonal antibodies to the 6-histidine epitope (Sigma, # H1029, dilution 1: 2000) were used. To the right is the position of the bands in the Precision Plus Dual Color Protein Standard (# 161-0374 Bio-Rad) protein molecular weight marker.

FIG. 5 shows a diagram of a vitro recombination test using the model plasmid pBl-Lox-PSMC-Lox-F3. As a result of the action of Cre from a circular plasmid with a length of 4743 bp. (S) a DNA fragment flanked by two unidirectional LoxP sites is excised. As a result of recombination, two circular recombination products with lengths of 3183 (R1) and 1560 (R2) bp are formed. The size of the linear DNA fragments formed after treatment of the substrate and the reaction products with EcoRI restriction endonuclease are shown under the scheme.

FIG. 6 shows the results of an in vitro recombination test using the model plasmid pBl-Lox-PSMC-Lox-F3. The recombination reaction products were analyzed on a 1% TAE-agarose gel. Lane "Cre-" - the substrate was incubated without the addition of 6His-S-NLS-Cre; lane "Cre +" - the substrate was incubated in the presence of 1 μM 6His-S-NLS-Cre. S - DNA fragments formed upon hydrolysis of the substrate with restriction endonuclease EcoRI. R1 and R2 are DNA fragments formed upon hydrolysis of the reaction products with restriction endonuclease EcoRI. The sizes of the reaction products are indicated on the left. The fragment sizes of a DNA molecular weight marker (O'GeneRuler 1kb DNA Ladder, ThermoFisher Scientific) are shown to the right.

Examples of implementation of the present invention

Example 1.

Preparation of a recombinant plasmid encoding a Cre protein cDNA fused with 6-histidine and S-epitopes

For the production of Cre protein in a bacterial expression system, a recombinant plasmid construct is obtained using genetic engineering methods. For this Cre recombinase cDNA was amplified by PCR on the template plasmid pBluescriptSK II (+) - TERT- Surv-Cre-3DNMT1 (Bezborodova et al, 2018.) With oligonucleotide primers 5-a gaattc gagctccccaagaagaagaggaag-3 and 5-a gtcgac ctaatcgccatcttccag- 3 containing the recognition sites of restriction endonucleases EcoRI and SalI, respectively (in bold). The amplification product was analyzed by electrophoresis in 1% agarose gel, purified from the agarose gel using the QiaexII reagent kit (Qiagen) and cloned into the pJET1 vector (ThermoFisher Scientific) to obtain the pJET1-Cre vector. Next, the complete nucleotide sequence of the amplified fragment is determined using Sanger sequencing.

Further, based on the vector pET-32a (+) (Merck), a modified vector pET32v11 is obtained. For this, a NotI-XhoI fragment containing stop codons is cloned from the pGEX-6P1 vector (GE Healthcare) into the pET-32a (+) vector at the NotI-XhoI sites. A DNA fragment (5-ccatgg gcagatctgaaaacctgtactttcagagc ggatcc -3) containing a TEV protease cleavage site is cloned into the resulting vector at the NcoI-BamHI sites (highlighted in bold). Next, the NdeI-NdeI fragment carrying the thioredoxin coding sequence is removed from the resulting vector, and the vector pET32v11 (SEQ ID NO 1) is obtained.

In vector pET32v11, the cleavage site for TEV protease is located so that after purification of the recombinant protein by metal affinity chromatography using TEV protease, the N-terminal fragment of the purified protein, including the 6-histidine and S-epitopes, can be cleaved if necessary.

Next, the EcoRI-SalI fragment containing the Cre cDNA is cloned from the pJET1-Cre vector into the pET32v11 plasmid vector at the EcoRI-SalI sites so that the Cre cDNA is in frame with the N-terminal 6-histidine epitope. The resulting recombinant plasmid was named pET32v11-Cre.

The resulting plasmid construct has a size of 6648 bp (SEQ ID NO 2). The genetic map of the plasmid pET32v11-Cre is shown in FIG. 1. The 6His-S-NLS-Cre recombinant fusion protein encoded by this plasmid has a calculated molecular weight of 46.2 kDa and consists of 411 amino acid residues (SEQ ID NO 3).

Example 2.

Procedure for obtaining a bacterial strain E.coli BL21 (DE3) / pET32v11-Cre producing recombinant protein 6-His-S-NLS-Cre

Plasmid vector pET32v11-Cre is transformed into a strainE.coli BL21 (DE3) using the standard bacterial transformation technique (Titus, 1991) and plated on LB agar (0.171 M NaCl, 1% tryptone, 0.5% yeast extract, 1.5% agar) containing the selective antibiotic ampicillin at a concentration of 50 μg / ml and grown for 16 hours at a temperature of + 37 ° C. Single colonies of bacteria grown on a selective medium are streaked onto a fresh selective dish and grown for 16 hours at a temperature of + 37 ° C. A single colony of bacteria is taken from a Petri dish with a bacterial loop and inoculated with 5 ml of liquid LB growth medium containing ampicillin at a concentration of 100 μg / ml and grown for 16 hours at a temperature of + 37 ° C and constant stirring. 200 μl of an overnight culture is transferred into 2 ml of LB medium containing ampicillin at a concentration of 100 μg / ml and grown for 2 hours at a temperature of + 37 ° C. CellsE.coli precipitated by centrifugation, the supernatant is removed, the precipitate is resuspended in 500 μl of freezing medium (liquid LB medium containing 15% glycerol). The resulting producer strain is stored at -80 ° C.

Example 3.

Procedure for obtaining recombinant protein 6His-S-NLS-Cre in a bacterial expression system

The cells of the E. coli BL21 (DE3) strain are transformed with the pET32v11-Cre plasmid and plated on a Petri dish with LB agar containing 0.5% glucose and 50 μg / ml ampicillin. Several bacterial colonies are washed off the Petri dish and resuspended in Terrific Broth medium warmed up to + 37 ° C (TB; 17 mM KH ₂ PO ₄ , 72 mM K ₂ HPO ₄ , 5 mM MgSO ₄ , 1.2% tryptone, 2.4% yeast extract , 0.5% glycerol) and inoculate two 2-liter flasks containing 500 ml of liquid TB medium and ampicillin at a concentration of 50 μg / ml. The bacteria grow with constant stirring and a temperature of + 37 ° C for 3-5 hours until an optical density of 0.4 at a wavelength of 600 nm is reached. Next, the flasks are cooled to + 18 ° C and the expression of the recombinant protein is induced by adding IPTG to a final concentration of 0.8 mM, ampicillin is added to a concentration of 50 μg / ml, and bacteria are grown at a temperature of + 18 ° C for 16 hours with constant stirring (220 rpm / min).

The cells are precipitated by centrifugation at 5000 rpm and a temperature of + 4 ° C for 20 minutes, the supernatant is removed, and the precipitate is washed using 50 ml of phosphate-buffered saline (0.01 M sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, pH = 7.4 ), pre-cooled to + 4 ° C, and then the cells are precipitated by centrifugation at 5000 rpm and a temperature of + 4 ° C for 20 minutes. The supernatant is removed, the cell mass is frozen in liquid nitrogen and stored at -80 ° C until the protein purification step.

The bacterial cell suspension is slowly thawed on ice, resuspended in 30 ml of lysis buffer (30 mM HEPES pH = 7.4, 500 mM NaCl, 10 mM KCl, 1 mM MgCl ₂ , 10 mM imidazole, 0.1% NP-40, 5% glycerol, 1 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 1x cocktail of protease VII inhibitors (Calbiochem) and disrupted with LV1 homogenizer (Microfluidics).

The resulting lysate of bacterial cells is centrifuged at 15000 rpm and a temperature of + 4 ° C for 60 minutes, the clarified lysate is collected. Protein purification is carried out on an Acta Pure medium pressure chromatograph (GE Healthcare). The clarified lysate is applied to a chromatographic column packed with 5 ml Ni-NTA agarose metal chelate affinity support (Qiagen) equilibrated with lysis buffer. The column is washed with buffer (30 mM HEPES pH = 7.4, 500 mM NaCl, 30 mM imidazole, 0.1% NP-40, 1 mM β-mercaptoethanol). Proteins bound to the column are eluted using a buffer (30 mM HEPES pH = 7.4, 500 mM NaCl, 300 mM imidazole, 1 mM β-mercaptoethanol). Collect fractions with a maximum absorption peak at a wavelength of 280 nm.

Fractions eluted from Ni-NTA columns are pooled, diluted twice with buffer (10 mM NaKPO ₄ pH = 6.8, 10 mM β-mercaptoethanol) and loaded onto a Mono S 5/50 cation exchange column (GE Healthcare) equilibrated with buffer (10 mM NaKPO ₄ pH = 6.8, 250 mM NaCl, 10 mM β-mercaptoethanol). The column is washed with buffer (10 mM NaKPO ₄ pH = 6.8, 250 mM NaCl, 10 mM β-mercaptoethanol). The proteins bound to the column are eluted using a salt gradient from 500 mM NaCl to 850 mM NaCl in 10 mM NaKPO ₄ buffer pH = 6.8, 10 mM β-mercaptoethanol. Collect fractions with a volume of 1 ml. Fractions with a maximum absorption peak at 280 nm were analyzed by SDS-PAGE on a 12% acrylamide gel (Fig. 2) as described (Laemmli, 1970). Recombinant protein 6His-S-NLS-Cre is efficiently eluted from the column at 700 mM NaCl. The result of the analysis of the fractions collected during the purification of the 6His-S-NLS-Cre recombinant protein is shown in Fig. 2. It can be seen that a protein with a molecular weight of about 45 kDa is eluted from the Mono S column, which is in line with the expected. Fractions descended from the Mono S column and containing the most pure recombinant protein 6His-S-NLS-Cre are dialyzed against 20 mM sodium acetate pH = 5.0, 40 mM NaCl, 2 mM DTT. Freeze in liquid nitrogen in aliquots and store at -80 ° C. To test the activity of the 6His-S-NLS-Cre protein, an aliquot is thawed, a functional test for in vitro recombination is performed (Figs. 5 and 6) and a sample of the recombinant protein is analyzed using SDS-PAGE and Western blotting (Figs. 3 and 4).

Example 4 .

Procedure for determining the identity and concentration of the purified recombinant protein 6His-S-NLS-Cre

The concentration of the purified recombinant protein 6His-S-NLS-Cre is determined spectrophotometrically by measuring the absorption of the protein sample at a wavelength of 280 nm in comparison with the dialysis buffer on a NanoDrop ND-8000 spectrophotometer (ThermoFisher Scientific). To calculate the protein concentration, the molar extinction coefficient for the 6His-S-NLS-Cre protein is used, calculated from its amino acid sequence, and is 50550. In this case, 1 unit of optical density at a wavelength of 280 nm corresponds to a protein concentration of 0.91 mg / ml.

The identity of the purified recombinant protein 6His-S-NLS-Cre is determined using SDS-PAGE in 10% acrylamide gel and Western blotting. Coli BL21 (DE3) / pET32v11-Cre bacterial cell lysates are prepared before the induction of recombinant protein synthesis (0 h) and after cultivation of the strain cells in a medium containing an IPTG inducer at a concentration of 1 mM for 3 hours at + 37 ° C. Samples were prepared containing 0.85 and 1.7 μg of purified 6His-S-NLS-Cre protein. Protein electrophoresis is carried out in 10% acrylamide gel and the gel is stained with Coomassie dye. FIG. 3 shows that the purified recombinant protein 6-His-S-NLS-Cre (lanes "0.85 μg" and "1.7 μg") migrates in the gel between the 37 and 50 kDa molecular weight marker bands, which is close to the expected molecular weight of the protein 6His-S-NLS-Cre at 46.2 kDa. The purity of the purified protein (tracks "0.85 µg" and "1.7 µg") is more than 90%. The amount of 6-His-S-NLS-Cre protein detected in the gel and the absence of intense bands of low molecular weight in the gel, which could indicate the degradation of the 6-His-S-NLS-Cre protein, indicate the stability of the obtained recombinant protein upon repeated freezing. and thawing.

Western blot analysis is performed to confirm the identity of the purified protein. Electrophoretic separation of the above samples is carried out in 10% acrylamide gel, proteins are transferred from the gel to a PVDF membrane (Millipore) using an SV20-SDB electrotransfer device (Sigma). The membrane is blocked in TBS-T buffer (50 mM Tris-HCl pH = 8.0, 138 mM NaCl, 2.7 mM KCl, 0.05% Tween-20) containing 5% skim milk powder (TBS-T5%). Next, the membrane is incubated for 16 hours at + 4 ° C in TBS-T5% buffer with mouse monoclonal antibodies to the 6 histidine epitope (Sigma, # H1029) at a dilution of 1: 2000. The membrane was washed three times in TBS-T buffer and incubated for 1 hour at room temperature with secondary antibodies against mouse immunoglobulins conjugated to horseradish peroxidase (GE Healthcare, # NA931V) at a dilution of 1: 5000. The membrane is washed three times in TBS-T buffer, and bound antibodies are detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Chemiluminescence is detected using a ChemiDoc XRS gel documentation system (Bio-Rad). FIG. 4 shows the result of Western blot analysis. It can be seen that using antibodies to the 6-histidine epitope, bands with a molecular weight slightly below 50 kDa (lanes "3 h", "0.85 μg" and "1.7 μg") are detected, which corresponds to the expected molecular weight of the 6His-S-NLS protein -Cre at 46.2 kDa. It is important to note that an intense immunoreactive band of the expected molecular weight is present only in a lysate sample of E. coli BL21 (DE3) / pET32v11-Cre cells after induction of recombinant protein synthesis (lane 3 h), but not in an un-induced cell lysate (lane 0 h "), in which only a weak immunoreactive band is detected, which reflects protein synthesis in the absence of induction of expression due to the" leakage "of the lacUV5 promoter in the E. coli genome, which controls the expression of T7 RNA polymerase. These results confirm the identity of the purified recombinant protein. In the "0.85 μg" and "1.7 μg" lanes, bands with a molecular weight of less than 37 kDa are detected, which are either degradation products of the purified protein, or products of premature termination of protein synthesis in E. coli cells. The intensity of the staining of these bands is due to the high concentration of protein in the analyzed samples. As seen in FIG. 3, the intensity of the low-molecular-weight bands is significantly weaker than the band in the gel corresponding to the purified Cre protein, which indicates the stability of the 6His-S-NLS-Cre protein.

Example 5.

In vitro site-specific recombination test

To carry out the in vitro recombination reaction, the pBl-Lox-PSMC-Lox-F3 plasmid with a size of 4347 bp is used as a substrate. (SEQ ID NO 4), in which the nucleotide sequence of the transcriptional terminator from the human PSMC5 gene is flanked by two unidirectional LoxP sites. As a result of the action of Cre recombinase, a DNA fragment is excised from the circular plasmid substrate with the formation of two circular recombination products of 3183 bp in size. and 1560 bp. (Fig. 5). The reaction products and substrate are linearized using restriction endonuclease EcoRI and analyzed by electrophoresis in 1% agarose gel. Upon hydrolysis of the substrate with the restriction endonuclease EcoRI, DNA fragments of 4298, 313, 114 and 18 bp in length are formed, designated S in FIG. 6, with the exception of a fragment of 18 bp, which is not detected in the gel; during the hydrolysis of the reaction products, fragments of 3051, 114, and 18 bp in length are formed. (R1) and 1560 bp (R2). A schematic diagram of the in vitro site-specific recombination test is shown in FIG. 5.

To carry out the recombination reaction, a mixture of 40 μl volume is prepared, containing 50 mM Tris-HCl pH = 7.5, 10 mM MgCl ₂ , 30 mM NaCl, 1 μg of plasmid DNA, 1 μM 6-His-S-NLS-Cre. The reaction is incubated at + 37 ° C for 30 minutes. Further, to inactivate the Cre recombinase, the reaction is incubated for 10 min at + 70 ° C. After that, 5 μl of 10-fold buffer "Orange" (ThermoFisher Scientific "), 4 μl of water and 1 μl (10 units of activity) of EcoRI restriction endonuclease (ThermoFisher Scientific") are added to the reaction. The reaction is incubated at + 37 ° C for 1 hour. Add 1 μl of proteinase K (20 μg / μl, "Sigma-Aldrich") and incubate the reaction for 10 minutes at + 56 ° C for the proteolysis of Cre and the release of the reaction products. Analyze the reaction products in 1% agarose gel.

FIG. 6 shows the result of the site-specific recombination test. Upon incubation of plasmid DNA pBl-Lox-PSMC-Lox-F3 with recombinant protein 6-His-S-NLS-Cre (sample Cre +) at + 37 ° C, reaction products (R1 and R2) of the expected size are formed. When the plasmid is incubated without protein addition, only fragments of the original substrate (S) with a length of 4298, 313, and 114 bp are detected. are formed during hydrolysis of both the substrate and the reaction products, it is the appearance of reaction products with a length of 3051 bp. (R1) and 1560 bp (R2) indicates the passage of Cre-mediated recombination.

The main characteristics of cells (according to the passport of the strain)

1. Generic and specific name of the host (recipient) strain: Escherichia coli

2. Strain number or name: BL21 (DE3) / pET32v11-Cre

3. Method of obtaining the strain: obtained by transformation.

4. Field of application of the strain: production of recombinant protein 6His-S-NLS-Cre

5. Product synthesized by the strain: 6His-S-NLS-Cre protein

6. Activity (productivity) of the strain, as well as other production indicators: 17 μg of protein per 1 ml of medium

7. Method for determining the activity of the strain indicating the method: in vitro test for recombination

8. Method, conditions and composition of media for long-term storage of the strain: medium LB + 15% glycerol in ampoules at -80 ° C

9. Method, conditions and composition of media for propagation of the strain: LB medium containing 50 μg / ml ampicillin

10. Group at risk of strain: I

11. Genetic characteristics of the strain:

a. The genotype of the host strain (mutations, deletions, inversions, the presence of plasmids or prophages, resistance (sensitivity) to antibiotics, phages, etc., other genetic features): genotype of the strain genotype of the strain BL21 (DE3): F ^- ompT gal dcm lon hsdS _B ( r _B ^- m _B ^- ) λ (DE3 [ lacI lacUV5 - T7p07 ind1 sam7 nin5 ]) [ malB ⁺ ] _K-12 (λ ^S )

b. Description of the recombinant plasmid

Plasmid name: pET32v11-Cre

Plasmid pET32v11-Cre size: 6648 bp

The complete nucleotide sequence of the plasmid: SEQ ID NO: 2.

v. Information about the vector on the basis of which the plasmid was constructed:

Vector name; pET-32a (+)

The origin of the vector and its main genetic elements;

Vector pET-32a (+) contains:

- gene for ampicillin resistance (β-lactamase)

- gene lac repressor ( lacI ) from E. coli

- the origin of replication from the ColE1 / pBR322 plasmids from E. coli .

- the origin of replication from bacteriophage f1

- the rop gene from E. coli , encoding a protein that regulates the copy number of the plasmid

- coding sequence of the trxA thioredoxin gene from E. coli

d. Information about cloned DNA

Donor species: Escherichia virus P1

General information about the cloned fragment, size and genes included in its composition; the plasmid contains the cDNA of the Cre gene, 1026 bp in length, encoded by the P1 bacteriophage genome (Genebank Acc .: NC_005856.1)

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--->

<110> Federal State Budgetary Institution of Science Institute

gene biology of the Russian Academy of Sciences (Institute of Gene Biology Russian

Academy of Sciences)

<120> Escherichia coli BL21 (DE3) / pET32v11-Cre cell strain producing

site-specific recombinase Cre

<160> 4

<210> 1

<211> 5596

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pET32v11

<400> 1

gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct 60

ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc 120

attcttctgg tctggtgcca cgcggttctg gtatgaaaga aaccgctgct gctaaattcg 180

aacgccagca catggacagc ccagatctgg gtaccgacga cgacgacaag gccatgggca 240

gatctgaaaa cctgtacttt cagagcggat ccccggaatt cccgggtcga ctcgatcgag 300

cggccgcatc gtgactgact gacgatctcg agcaccacca ccaccaccac tgagatccgg 360

ctgctaacaa agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag 420

cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta 480

tatccggatt ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt 540

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 600

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 660

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 720

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 780

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 840

ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 900

gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta caatttcagg 960

tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 1020

aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 1080

gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 1140

ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 1200

gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 1260

tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 1320

attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 1380

tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 1440

agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 1500

aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 1560

tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 1620

cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 1680

tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 1740

tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 1800

tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 1860

tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 1920

aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 1980

gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 2040

tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 2100

aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 2160

aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 2220

tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 2280

gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 2340

cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 2400

acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 2460

cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 2520

cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 2580

aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 2640

gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 2700

atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 2760

tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 2820

gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 2880

agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg 2940

catatatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca 3000

ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg 3060

acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3120

ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc 3180

ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt 3240

ccagctcgtt gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt 3300

taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca 3360

tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg 3420

aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg 3480

accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc 3540

cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg 3600

acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc 3660

aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat 3720

tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga 3780

tcatgcgcac ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt 3840

tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa 3900

gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga 3960

gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga 4020

cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca 4080

tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac 4140

tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4200

cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg 4260

ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg 4320

ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat 4380

gagctgtctt cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg 4440

gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca 4500

gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc 4560

cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag 4620

ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc 4680

tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa 4740

ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg 4800

caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca 4860

ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt 4920

tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg 4980

acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac 5040

tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc 5100

gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa 5160

acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca 5220

ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg 5280

cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa 5340

gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa 5400

ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca 5460

agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata 5520

ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag 5580

gatcgagatc gatctc 5596

<210> 2

<211> 6648

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pET32v11-Cre:

gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa caattcccct 60

ctagaaataa ttttgtttaa ctttaagaag gagatataca tatgcaccat catcatcatc 120

attcttctgg tctggtgcca cgcggttctg gtatgaaaga aaccgctgct gctaaattcg 180

aacgccagca catggacagc ccagatctgg gtaccgacga cgacgacaag gccatgggca 240

gatctgaaaa cctgtacttt cagagcggat ccccggaatt cgagctcccc aagaaga 300

ggaaggtgtc caatttactg accgtacacc aaaatttgcc tgcattaccg gtcgatgcaa 360

cgagtgatga ggttcgcaag aacctgatgg acatgttcag ggatcgccag gcgttttctg 420

agcatacctg gaaaatgcta ctgtccgttt gccggtcgtg ggcggcatgg tgcaagttga 480

ataaccggaa atggtttccc gcagaacctg aagatgttcg cgattatctt ctatatcttc 540

aggcgcgcgg tctggcagta aaaactatcc agcaacattt gggccagcta aacatgcttc 600

atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc tgtttcactg gttatgcggc 660

ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa acaggctcta gcgttcgaac 720

gcactgattt cgaccaggtt cgttcactca tggaaaatag cgatcgctgc caggatatac 780

gtaatctggc atttctgggg attgcttata acaccctgtt acgtatagcc gaaattgcca 840

ggatcagggt taaagatatc tcacgtactg acggtgggag aatgttaatc catattggca 900

gaacgaaaac gctggttagc accgcaggtg tagagaaggc acttagcctg ggggtaacta 960

aactggtcga gcgatggatt tccgtctctg gtgtagctga tgatccgaat aactacctgt 1020

tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc caccagccag ctatcaactc 1080

gcgccctgga agggattttt gaagcaactc atcgattgat ttacggcgct aaggatgact 1140

ctggtcagag atacctggcc tggtctggac acagtgcccg tgtcggagcc gcgcgagata 1200

tggcccgcgc tggagtttca ataccggaga tcatgcaagc tggtggctgg accaatgtaa 1260

atattgtcat gaactatatc cgtaacctgg atagtgaaac aggggcaatg gtgcgcctgc 1320

tggaagatgg cgattaggtc gactcgatcg agcggccgca tcgtgactga ctgacgatct 1380

cgagcaccac caccaccacc actgagatcc ggctgctaac aaagcccgaa aggaagctga 1440

gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt 1500

cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg ggacgcgccc 1560

tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 1620

gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 1680

ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 1740

cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 1800

tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 1860

ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 1920

ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 1980

tttaacaaaa tattaacgtt tacaatttca ggtggcactt ttcggggaaa tgtgcgcgga 2040

acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 2100

ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 2160

gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 2220

ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 2280

gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 2340

agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag 2400

caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca 2460

gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg 2520

agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc 2580

gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg 2640

aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg 2700

ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac 2760

tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg 2820

tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg 2880

gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact 2940

atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa 3000

ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt 3060

aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag 3120

ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct 3180

ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt 3240

tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg 3300

cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct 3360

gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc 3420

gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg 3480

tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa 3540

ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg 3600

gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg 3660

ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga 3720

tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt 3780

ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct 3840

gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga 3900

acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt 3960

ctccttacgc atctgtgcgg tatttcacac cgcatatatg gtgcactctc agtacaatct 4020

gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg actgggtcat 4080

ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 4140

ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 4200

accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt ggtcgtgaag 4260

cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct ccagaagcgt 4320

taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct gtttggtcac 4380

tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga tgaaacgaga 4440

gaggatgctc acgatacggg ttactgatga tgaacatgcc cggttactgg aacgttgtga 4500

gggtaaacaa ctggcggtat ggatgcggcg ggaccagaga aaaatcactc agggtcaatg 4560

ccagcgcttc gttaatacag atgtaggtgt tccacagggt agccagcagc atcctgcgat 4620

gcagatccgg aacataatgg tgcagggcgc tgacttccgc gtttccagac tttacgaaac 4680

acggaaaccg aagaccattc atgttgttgc tcaggtcgca gacgttttgc agcagcagtc 4740

gcttcacgtt cgctcgcgta tcggtgattc attctgctaa ccagtaaggc aaccccgcca 4800

gcctagccgg gtcctcaacg acaggagcac gatcatgcgc acccgtgggg ccgccatgcc 4860

ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg 4920

agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca 4980

gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg 5040

catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa 5100

ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt 5160

gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc 5220

gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg 5280

ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct 5340

ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct 5400

gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca 5460

ctaccgagat gtccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca 5520

gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt 5580

gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct 5640

gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag 5700

aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca 5760

cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag 5820

agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct 5880

ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca 5940

ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac 6000

ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca 6060

gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc 6120

ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag 6180

aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact 6240

ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg 6300

ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga 6360

cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg 6420

agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc 6480

acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc 6540

cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg 6600

gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tcgatctc 6648

<210> 3

<211> 411

<212> PRT

<213> artificial sequence

<220>

<223> Protein 6-His-S-NLS-Cre

<400> 3

Met His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 16

1 5 10 15

Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 32

20 25 30

Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Gly Arg Ser 48

35 40 45

Glu Asn Leu Tyr Phe Gln Ser Gly Ser Pro Glu Phe Glu Leu Pro Lys 64

50 55 60

Lys Lys Arg Lys Val Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro 80

65 70 75 80

Ala Leu Pro Val Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met 96

85 90 95

Asp Met Phe Arg Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met 112

100 105 110

Leu Leu Ser Val Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn 128

115 120 125

Arg Lys Trp Phe Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu 144

130 135 140

Tyr Leu Gln Ala Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu 160

145 150 155 160

Gly Gln Leu Asn Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser 176

165 170 175

Asp Ser Asn Ala Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn 192

180 185 190

Val Asp Ala Gly Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr 208

195 200 205

Asp Phe Asp Gln Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln 224

210 215 220

Asp Ile Arg Asn Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu 240

225 230 235 240

Arg Ile Ala Glu Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr 256

245 250 255

Asp Gly Gly Arg Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val 272

260 265 270

Ser Thr Ala Gly Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu 288

275 280 285

Val Glu Arg Trp Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn 304

290 295 300

Tyr Leu Phe Cys Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala 320

305 310 315 320

Thr Ser Gln Leu Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr 336

325 330 335

His Arg Leu Ile Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu 352

340 345 350

Ala Trp Ser Gly His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala 368

355 360 365

Arg Ala Gly Val Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr 384

370 375 380

Asn Val Asn Ile Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr 400

385 390 395 400

Gly Ala Met Val Arg Leu Leu Glu Asp Gly Asp 411

405 410

<210> 4

<211> 4743

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pBl-Lox-PSMC-Lox-F3

<440> 4

ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60

attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120

gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180

caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240

ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300

cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360

agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420

cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtacccc 660

acccgggtgg aaaatcgatg ggcccgcggc cgctctagaa gtactctcga gtaaactcct 720

cttcagacct aataacttcg tatagcatac attatacgaa gttatattaa gggttattga 780

atattatcgg gaattaattc tttggatcca ctagtgtcga ggtcaacggc cgcatgtatg 840

ccctgcgaga acggcgagtc catgtcactc aggaggactt tgagatggca gtagccaagg 900

tataggcctc catctttgtg cctttgccag tggtggctct ggggcagtgg gctagggcat 960

gtgtgtgttc gtaacttaat gttcattctc tctcccaccc ctaggtcatg cagaaggaca 1020

gtgagaaaaa catgtccatc aagaaattat ggaagtgagt ggacagcctt tgtgtgtatc 1080

tctccaataa agctctgtgg gccaagtcct ctaggactcc agtctgttaa tgagggctgt 1140

gctgttcaag gaccaggtcc aggcaccacc aatagacaaa aggatttatt tggaaatttc 1200

caaacctgcc agatgtggca ctcgccaagc cctaggccca ccctcctcac caagctccaa 1260

agggcaacac cagtcctccc agcctcccca ttcaccttac cctggcctcc acatgcacat 1320

accccatacc tcaggccatg ctagagaact ggagtgtccc ctccccggcc caagccgctg 1380

gagaggcagc cctctggcat cccaggagat gatggcggcc agagccgctt gcatagattg 1440

aggaaagaaa agaaggaggc acctaacagg ctccctgaaa acccccaact tacccctgta 1500

caaaaaaagt ggctcccaca tagaaaactt gctcccaaaa tagtgcaaat acccaaagga 1560

aaggaaaaaa ccagcagcaa ggctgggctt gtaggaatgg caaagaactt tggtttagaa 1620

aggctaggaa gagtctgggc tgcacctcct cttcctagcc cagctcccca gagccagctc 1680

tgaccctcgc cccaggggga gtctgtaaac atgcaaaccc agcagccttt ggttcggaaa 1740

tgtcttacaa tgtcaaagca cagcctccag cagtcctact tgggcctgcc tgcagggggg 1800

cagaggaggc atctgctgaa cccaattaaa gccaatgcaa aaagtataag gctttgggga 1860

ccaagcaaca aggaatccta tcactacatt tataagacta aagctggaga gtagagggtg 1920

acagcagaca ctcctcaccc cagcctacgt gtctgcggcc ccagcaagga cccagagggt 1980

ggtctccctc caggctccag ggctgggaag aaagatccct gagcagttag gtcaggcgaa 2040

ttcccagcac ctgttccagt tcctgccttc gctgctgcac ctggagaagg gagaaaccaa 2100

gtggctcagg cctttgctac tcacggccaa aggagggaaa acagggcaga gcctcacctt 2160

ggcaaagatg tgcctgccta ctgcttcctg ggcccagggc tggtggtaga aagcagctcg 2220

tctctccggc cgttgacggt atcgataagc ttgatctaga actagtggat ctaaactcct 2280

cttcagacct aataacttcg tatagcatac attatacgaa gttatattaa gggttattga 2340

atattatcgg gaattcgata tcaagcttat cgataccgtc gacggtatcg ataagcttga 2400

agttcctata ctatttgaag aataggaact tcggaatagg aacttcaaga tccccctggc 2460

gaaggaattc ctgcagccca tcgaattcct gcagcccggg ggatccacta gttctagagc 2520

ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct 2580

tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 2640

acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 2700

tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 2760

tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 2820

cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 2880

actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 2940

gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 3000

ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 3060

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 3120

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 3180

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 3240

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 3300

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 3360

ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 3420

acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 3480

gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 3540

ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 3600

tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 3660

gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 3720

tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 3780

ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 3840

taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 3900

cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 3960

gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 4020

gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 4080

tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 4140

gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 4200

ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 4260

ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 4320

cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 4380

ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 4440

gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 4500

ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 4560

ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 4620

tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 4680

ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 4740

cac 4743

Free text sequence listing

<110> Federal State Budgetary Institution of Science Institute of Biology

gene of the Russian Academy of Sciences (Institute of Gene Biology Russian Academy of

Sciences)

<120> Escherichia coli BL21 (DE3) / pET32v11-Cre cell strain producing

site-specific recombinase Cre

<160> 4

<210> 1

<211> 5596

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pET32v11:

102-311 bp -> straight chain, 210 bp; reading frame encoding chimeric

a protein comprising a 6-histidine epitope, a thrombin cleavage site, an S-epitope,

cleavage sites by enterokinase and TEV protease.

1088-1945 bp -> straight chain, 858 bp; ampicillin resistance gene

(β-lactamase) 4140-5219 nt -> reverse chain, 1080 bp; lac repressor gene

(lacI) from E. coli 3137-3328 bp -> reverse circuit, 192 bp; the rop gene from E. coli,

coding protein,

copy number regulating plasmid

<210> 2

<211> 648

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pET32v11-Cre:

102-1334 bp -> straight chain, 1233 bp; reading frame encoding

recombinant protein 6-His-S-NLS-Cre

2140-2997 bp -> straight chain, 858 bp; ampicillin resistance gene

(β-lactamase)

5192-6271 bp -> return circuit, 1080 bp; lac repressor gene (lacI) from

E.coli

4189-4380 bp -> reverse circuit, 192 bp; coli rop gene encoding

plasmid copy regulation protein

<210> 3

<211> 411

<212> PRT

<213> artificial sequence

<220>

<223> Protein 6-His-S-NLS-Cre:

2-7: 6-histidine epitope (HHHHHH)

11-16: Thrombin cleavage site (LVPRGS)

19-33: S epitope from pancreatic RNase A (KETAAAKFERQHMDS)

39-43: Enterokinase cleavage site (DDDDK)

49-55: TEV protease cleavage site (ENLYFQS)

63-69: Signal of nuclear localization from the large T antigen of the virus SV40, NLS

(PKKKRKV)

70-411: Amino acid sequence of Cre protein without starting methionine

(2-343 a.s.)

<210> 4

<211> 4743

<212> DNA

<213> artificial sequence

<220>

<223> Plasmid construct pBl-Lox-PSMC-Lox-F3:

3755-4612 bp -> straight chain, 858 bp; ampicillin resistance gene

(β-lactamase)

<---

Claims (4)

1. Recombinant plasmid pET32v11-Cre, having the sequence SEQ ID NO: 2 and providing the synthesis of the recombinant protein 6His-S-NLS-Cre, having the sequence SEQ ID NO: 3, in Escherichia coli cells , due to the location of the Cre recombinase gene in 3 ' -regions relative to the T7 promoter and lac operator and in the 5'-region relative to the T7 terminator, and obtained by cloning the Cre recombinase cDNA into the pET32v11 vector having the sequence SEQ ID NO: 1, so that the Cre recombinase cDNA with the N-terminal NLS is in the same reading frame with the 6-histidine and S-epitopes and amino acid sequences of the cleavage sites of thrombin, enterokinase and TEV protease. 2. The Escherichia coli BL21 (DE3) / pET32v11-Cre cell strain, producing the recombinant 6His-S-NLS-Cre protein, which has the sequence SEQ ID NO: 3, and obtained by transforming the Escherichia coli BL21 (DE3) cell strain with the plasmid according to claim .one. 3. A method of producing a 6His-S-NLS-Cre protein having the sequence SEQ ID NO: 3, comprising the cultivation of a producer strain of Escherichia coli BL21 (DE3) / pET32v11-Cre according to claim 2, induction of the synthesis of a recombinant 6His-S-NLS protein -Cre having the sequence SEQ ID NO: 3, obtaining a cell lysate and purifying the 6His-S-NLS-Cre protein having the sequence SEQ ID NO: 3 using metal chelate affinity chromatography and cation exchange chromatography. 4. The method of claim 3, wherein the yield of the catalytically active Cre recombinase is about 17 μg per ml of culture.

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